L-Glutamate decarboxylase (GAD) is the enzyme involved in the synthesis of GABA, a major inhibitory neurotransmitter. There are two well characterized GAD isoforms in the brain, namely, GAD65 and GAD67 [(Erlander and Tobin, 1991)]. Recently, we have shown that soluble GAD (SGAD) is activated by dephosphorylation, mediated by calcineurin, and is inhibited by phosphorylation, mediated by PKA (Bao et al., 1994, 1995), whereas the membrane associated GAD (MGAD) is activated by protein phosphorylation which depends on the integrity of synaptic vesicles (Hsu et al., 1999). In addition, we have identified MGAD from synaptosomes as GAD65 (Hsu et al., 1999). Thus, GAD activity appears to be regulated also depending on the partition of GAD in soluble and membrane associated form. In addition, a detailed crystal structure of GAD is essential for understanding the regulation of GAD activity. Hence, we propose to perform the following studies: (1) To determine the three dimensional structure of GAD. This aspect of studies will be carried out in collaboration with Dr. F. Takusagawa (letter of collaboration is attached) who has recently determined the crystal structure of S- adenosylhomocysteine hydrolase (Hu et al., 1999). (2) To elucidate the mechanisms involved in anchoring GAD to synaptic vesicles with special focus on the role of heat shock cognate protein (HSC70) since our results indicate that GAD65, but not GAD67 forms complex with HSC70 (see Preliminary Results). (3) Characterization of GAD65 and GAD67 phosphorylation sites in terms of the identity of phosphoamino acids, the location (sequence), and the number of phosphorylation sites. In addition, site-directed mutagenesis will be used to determine which amino acid residues are important for phosphorylation and regulation of GAD activity. (4) To elucidate the physiological significance of GAD65 and GAD67 phosphorylation. (5) To address whether GAD65 and GAD67 activities are sensitive to intracellular GABA concentrations. The success of a large scale purification of recombinant human brain GAD65 and GAD67 and the availability of subtype specific antibodies for GAD65 and GAD67, ensure the high feasibility of this proposal.